Hepatitis C virus (HCV) is the etiologic agent of parenterally transmitted non-A, non-B viral hepatitis. Chronic infection puts individuals at risk for the development of cirrhosis, hepatocellular carcinoma, and liver failure, making chronic hepatitis C the leading indication for liver transplantation. While HCV-specific protease and polymerase inhibitors are showing promise in early clinical development, rapid emergence of resistance indicates that additional viral targets and combinations of antivirals will be needed for effective control. We propose to investigate the structure and function of the replicase complex, the central holoenzyme of HCV replication. Increased understanding of how the components of this complex come together, as well as clarifying the functions and mechanisms of each constituent, will facilitate the development of novel antiviral drugs. We will use genetic analyses complemented with biochemical studies to investigate protein-protein interactions within the replication complex. These studies will provide a context in which to build a structural understanding of the replicase. We then aim to elucidate the roles of two important but enigmatic viral proteins, the NS3 helicase and NS5A. These proteins, both potentially extremely valuable drug targets, have as-yet-unknown roles in RNA replication. We will investigate the mechanism of NS3 helicase activity and the roles of this enzyme in the viral life cycle using X-ray crystallography and complementary genetic approaches. We will pursue an atomic-resolution structure of full-length NS5A, as well as investigate its structure in complex with an RNA substrate. We will also attempt to develop cell-free replication assays for HCV; the current lack of such systems is a major roadblock to studies of this virus. Availability of a cell-free assay would allow us to relate structural, biochemical, and genetic data to specific steps of RNA synthesis. Through these studies, we hope to begin to understand mechanisms of HCV RNA replication and to uncover novel avenues for therapeutic intervention.